The ongoing dramatic rise in obesity and the lack of efficient intervention strategies to treat obesity demonstrates the need to better understand mechanisms to maintain energy homeostasis. Thermogenesis is necessary to maintain body temperature, but also affects energy expenditure and body weight. Thermogenesis controls heat production in peripheral tissues, particularly the brown adipose tissue (BAT). BAT thermogenesis is also functional in adult humans and despite controversial opinions on the importance of BAT function to control body weight, BAT size correlates negatively with body mass index and thus central regulators of BAT thermogenesis are potential targets for anti-obesity drugs. Leptin regulates body weight in part via thermoregulatory mechanisms including BAT heat production. Indeed, hypothalamic leptin responsive neurons (e.g. in the DMH/DHA) recapitulate known central thermoregulatory pathways, that we hypothesize to mediate food-intake independent body weight regulation. The proposed experiments investigate the role of DMH/DHA leptin action in thermogenesis and energy homeostasis, emphasizing physiological function, regulation of central and peripheral signaling pathways and neuronal connectivity of DMH/DHA neurons (inhibitory/excitatory LepRb inputs to the DMH/DHA). The proposed study of leptin receptor (LepRb) neurons in mice allows the use of several molecular biological state-of-the-art tools (cre/loxP technology, neuron specific tracing and reporter expression), that have not been used in the thermoregulation research field, yet. In Aim 1 we will study the physiological importance of thermoregulatory DMH/DHA leptin action on body weight control by using DMH/DHA specific leptin or LepRb antagonist injections, or by in vivo modulation of neuronal activity in DMH/DHA LepRb neurons using DREADD technology. Thermoregulatory leptin action in wildtype or leptin deficient ob/ob mice is monitored by temperature transmitters, VO2/CO2 exchange, protein markers of BAT function and effects on body weight as well as other peripheral tissues (e.g. muscle and white fat) are investigated for leptin induced changes (e.g fat oxidation, mitogenesis). In Aim 2 will identify inhibitory/excitatory (GABA-/glutamatergic inputs to the DMH/DHA (e.g. from the POA) and their response to leptin. Furthermore, we will study mice with loss of glutamate in LepRb DMH/DHA to test the physiological consequence for thermoregulation and body weight control. In Aim 3 we identify the neuronal connectivity of LepRb DMH/DHA neurons. We will use site and neuron- specific tracing techniques (stereotaxic injection of cre-inducible viral tracers) to visualize GABA-/glutamatergic projections (axonal EGFPf) and their 2nd order target neurons (transsynaptic tracing). In leptin deficient ob/ob mice with LepRb-driven cre/EGFP expression potential thermoregulatory defects (e.g. LepRb expression, neuronal projections) in POA & DMH/DHA neurons are investigated.